Methods involving macrophage tumor cell fusion hybrids

Abstract

Methods of identifying circulating macrophage-tumor cell fusion hybrids from a subject, methods of screening test compounds for inhibition of activity of in vitro and in vivo derived macrophage-tumor cell fusion hybrids, and methods of assessing the probability of survival of a human subject with pancreatic cancer past one year are disclosed.

1 . A method of identifying a circulating macrophage-tumor cell fusion hybrid from a subject, the method comprising:
 contacting a cell obtained from the blood of the subject with a first antibody that binds a macrophage marker, the first antibody comprising a first label and   contacting the cell with a second antibody that binds a tumor antigen, the second antibody comprising a second label, where the second label is distinguishable from the first label;   where a cell with detectable emission of a distinguishable signal from both the first label and the second label is identified as a circulating macrophage-tumor cell fusion hybrid.   
     
     
         2 . The method of  claim 1  where the first antibody binds CD45 and/or the second antibody binds a cytokeratin. 
     
     
         3 . The method of  claim 2  where the second antibody comprises a pan-cytokeratin antibody. 
     
     
         4 . The method of  claim 1  where the first label comprises a first fluorescent label that emits at a first wavelength and the second label comprises a second fluorescent label that emits at a second wavelength and where the first wavelength and the second wavelength can be distinguished. 
     
     
         5 . The method of  claim 4  further comprising isolating the circulating macrophage tumor cell fusion by fluorescence activated cell sorting (FACS). 
     
     
         6 . A method of screening a test compound for inhibition of one or more activities of an in vitro derived macrophage-tumor cell fusion hybrid, the method comprising:
 contacting a first sample comprising an in vitro derived macrophage-tumor cell fusion hybrid with the test compound;   contacting a second sample comprising a second in vitro derived macrophage-tumor cell fusion hybrid with a negative control;   measuring one or more of cellular proliferation, cellular migration, or metastasis, in the first sample and the second sample;   wherein a result showing less cellular proliferation, less cellular migration, and/or less metastasis, in the first sample relative to the second sample is an indication that the test compound inhibits the one or more activities of the in vitro derived macrophage-tumor cell fusion hybrid.   
     
     
         7 . The method of  claim 6  further comprising generating the in vitro derived macrophage-tumor cell fusion hybrid by contacting a macrophage with a tumor cell in a cell culture lacking an agent that promotes cell-cell fusion, where the macrophage expresses a first marker protein and the tumor cell expresses a second marker protein distinguishable from the first marker protein and purifying the macrophage-tumor cell fusion hybrid based on the expression of the first marker protein and the second marker protein. 
     
     
         8 . The method of  claim 7  where the first marker protein comprises a first fluorescent protein that fluoresces at a first wavelength and where the second marker protein comprises a second fluorescent protein that fluoresces at a second wavelength distinguishable from the first wavelength, and where purifying the macrophage-tumor cell fusion hybrid comprises flow cytometery. 
     
     
         9 . The method of  claim 8  where the first fluorescent protein comprises RFP, GFP, or YFP and where the second fluorescent protein comprises RFP, GFP, or YFP, provided that the first fluorescent protein and the second fluorescent protein are not both RFP, GFP, or YFP. 
     
     
         10 . The method of  claim 8  where measuring cellular proliferation comprises performing an MTS assay, where measuring cellular migration comprises performing a chemotaxis assay or a scratch wound assay, or where measuring metastasis comprises measuring liver metastases in an experimental animal after injection of the macrophage-tumor cell fusion hybrid into the spleen of the experimental animal. 
     
     
         11 . The method of  claim 7  where the macrophage and the tumor cell are each derived from a mouse. 
     
     
         12 . The method of  claim 11  where the macrophage is derived from a transgenic mouse that expresses a fluorescent protein in macrophages. 
     
     
         13 . The method of  claim 11  where the tumor cell is derived from a colon adenocarcinoma or a melanoma. 
     
     
         14 . The method of  claim 13  where the tumor cell comprises the MC38 line or the B16F10 line. 
     
     
         15 . A method of screening a test compound for inhibition of proliferation or metastasis of an in vivo derived macrophage-melanoma cell fusion hybrid or macrophage primary mammary tumor cell hybrid, the method comprising:
 injecting a melanoma cell line or a primary mammary tumor cell into a first mouse, where the melanoma cell line or primary mammary tumor cell expresses a first fluorescent protein, where the first mouse transgenically expresses a second fluorescent protein distinguishable from the first fluorescent protein, where the melanoma cell line or primary mammary tumor cell forms a tumor in the first mouse, and where the second fluorescent protein is expressed in macrophages;   removing the tumor from the first mouse;   purifying cells that express both the first fluorescent protein and the second fluorescent protein by flow cytometry to obtain in vivo derived macrophage-melanoma cell fusion hybrids or macrophage mammary tumor cell fusion hybrids;   injecting a portion of the purified cells that express both the first fluorescent protein and the second fluorescent protein into a second mouse;   administering the test compound to the second mouse;   injecting a portion of the purified cells that express both the first fluorescent protein and the second fluorescent protein into a third mouse; and   administering a negative control to the third mouse;   where a result showing less proliferation or metastasis of the cells that express both the first fluorescent protein and the second fluorescent protein in the second mouse relative to the third mouse is an indication that the test compound inhibits proliferation or metastasis of the in vivo derived macrophage-melanoma cell fusion hybrid or macrophage mammary tumor cell fusion hybrid.   
     
     
         16 . The method of  claim 15  where the second mouse and the third mouse are each injected with 50-10,000 cells that express both the first fluorescent protein and the second fluorescent protein. 
     
     
         17 . The method of  claim 15  comprising injecting the macrophage-melanoma cell fusion hybrids or macrophage-mammary tumor cell hybrids into a first cohort comprising a first plurality of mice and a second cohort comprising a second plurality of mice, injecting the test compound into the first cohort and the negative control into the second cohort. 
     
     
         18 . The method of  claim 15  comprising removing the tumor from the first mouse when the tumor is 1-2 cm in diameter. 
     
     
         19 . The method of  claim 15  where the melanoma cell line comprises a B16F10 cell line. 
     
     
         20 . The method of  claim 15  where the macrophage-melanoma cell fusion hybrids or macrophage mammary tumor cell fusion hybrids are injected into the second mouse, the third mouse, the first plurality of mice and/or the second plurality of mice intradermally. 
     
     
         21 . A method of assessing the probability of survival of a human subject with pancreatic cancer past one year, the method comprising:
 receiving a peripheral blood sample from the subject, the sample comprising mononuclear cells;   contacting the blood sample with an anti-CD45 antibody, the anti-CD45 antibody comprising a first fluorescent label;   contacting the blood sample with an anti-pan-cytokeratin antibody, the anti-pan cytokeratin antibody comprising a second fluorescent label distinguishable from the first fluorescent label;   contacting the blood sample with Hoescht stain;   counting the CD45+/cytokeratin+/Hoescht+ cells in at least a subset of the cells in the sample;   calculating the percentage of CD45+/cytokeratin+/Hoescht+ cells in the total cells in the subset;   
       where if more than 0.8% of the cells are CD45+/cytokeratin+/Hoescht+ relative to the total number of cells in the sample, the subject has a less than 12% chance of survival past one year. 
     
     
         22 . The method of  claim 21  further comprising adhering the peripheral blood sample to a glass substrate and fixing cells from the sample in 4% paraformaldehyde prior to contacting the peripheral blood sample with the anti-CD45 antibody and the anti-pan-cytokeratin antibody. 
     
     
         23 . The method of  claim 21  where the peripheral blood sample is a buffy coat fraction. 
     
     
         24 . The method of  claim 21  where the anti-CD45 antibody is a monoclonal antibody derived from the H130 clone and/or the anti-pan cytokeratin antibody is a monoclonal antibody derived from the C1-11 clone or a polyclonal antiserum. 
     
     
         25 . The method of  claim 21  where the subset of the cells comprises at least 2000 cells.